Evaluation of Hormonal Status

Introduction

This chapter reviews the assessment of hormonal status in the practice of reproductive endocrinology. It is acknowledged that the patient will often provide useful biologic information, such as changes induced by hypoestrogenism, skin changes with androgen excess, and galactorrhea in hyperprolactinemia. The clinically relevant symptoms and signs associated with various disorders will be discussed in other chapters in this text. Here we will describe various types of hormonal assays and dynamic tests used in the evaluation of reproductive disorders, as well as diagnostic radiographic techniques. We also offer several algorithms for the diagnosis of common clinical disorders with the use of hormonal assessments where necessary.

Hormone Assays

An understanding of the principles of all immunoassays is necessary to understand their usefulness and limitations.
There are several types of immunoassays as well as detection systems, some of which have been developed for rapidity and automation.
The gold standard for the measurement of steroid hormones, which may be at very low circulating levels, is with the use of mass spectrometry (MS).

Historical Aspects

The predominant assays used to measure protein and peptide hormones, as well as small-molecule hormones (e.g., steroids, thyroxine, vitamin D), in serum, plasma, and/or urine samples for over 50 years have been immunoassays. These assays have been widely used in both clinical diagnostic and research settings.

Historically, the first type of immunoassay utilized a radioactive marker; hence it was called a radioimmunoassay (RIA). The first RIA was developed in 1959 for insulin. In the next several years, this was followed by the development of RIAs for other proteins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as for peptides such as gonadotropin-releasing hormone (GnRH). The first steroid RIA was developed in 1969 for estradiol. It required preceding purification steps (organic solvent extraction and Celite or Sephadex column chromatography) prior to quantitation of estradiol, and it is often referred to as an indirect or conventional RIA. Soon afterward, indirect RIAs were developed for other steroids such as progesterone and testosterone, and for other small-molecule hormones. Although RIAs are seldom used now, they inaugurated a new era in endocrine research and clinical practice.

Due to the cumbersome and time-consuming nature of the RIA method, RIAs without a preceding purification step (direct RIAs) were developed in the late 1970s to measure small-molecule hormones. Around this time, a sandwich-type immunoassay, so-called because the assay principle involves “sandwiching” an analyte between two antibodies, was also developed for the measurement of protein and peptide hormones. This immunoassay also included a radioactive marker and was called an immunoradiometric assay (IRMA). Subsequently, the radioactive marker in both the direct RIA and IRMA methods was replaced with an enzymatic marker, and a competitive enzyme immunoassay (EIA) was developed for small-molecule hormones and a sandwich-type enzyme-linked immunosorbent assay (ELISA) for protein and peptide hormones. These assays are still widely used now, especially the ELISAs. Another major development in assay methodology that occurred in the early 1980s was the automation of hormone assays. This involved replacement of the radioactive label used in direct RIAs and in IRMAs with a chemiluminescent or fluorescent marker so that they could be used on an analyzer. Due to their greater sensitivity, chemiluminescent immunoassays prevailed and are widely used presently.

In recent years, there has been a growing trend to use mass spectrometry (MS) assays to measure steroid hormones. In the 1960s, gas chromatography-mass spectrometry (GC-MS) assays were already advanced enough to measure steroid hormones in serum/plasma and urine. However, this methodology was complex and costly, so RIAs became used much more widely. Liquid chromatography-mass spectrometry (LC-MS) assays are now the method of choice for use in the clinical laboratory for routine determination of steroids. MS assays are considered to be the “gold standard” for the measurement of steroid hormones.

Immunoassays

Although considerable attention has been paid to MS assays in the last several years, and they are considered to be the “gold standard” for hormone analysis, it is unrealistic to think that immunoassays will disappear. First of all, the chemiluminescent immunoassays for protein and peptide hormones on automated platforms or enzyme immunoassays performed manually provide useful measurements for clinical diagnosis and research. Assays for protein and peptide hormones using MS are not available for routine analysis. Second, although the direct immunoassays used to measure steroid hormones generally do not provide absolute values and lack sensitivity to measure low levels of certain hormones, for example, estradiol in postmenopausal women, they are used by many clinicians to determine serum hormone levels for diagnostic decisions. Also, in in vitro fertilization (IVF) settings, serum estradiol levels are supraphysiologic in response to ovarian stimulation, and the levels are overestimated. Nevertheless, immunoassays on an automated platform are the method of choice to measure these levels due to the rapid turnaround time of the assay. In addition to estradiol, the analyzer can be used to measure progesterone, FSH, LH, and human chorionic gonadotropin (hCG) rapidly. For those reasons, immunoassays are likely to be used for a substantial number of years.

As the term immunoassay implies, this method of measuring hormones involves an antigen-antibody reaction, where the antigen is the hormone to be measured and the antibody, which is prepared against the hormone, binds to the hormone. The hormone can be a protein, peptide, or small-molecule. There are two types of hormone immunoassay methods that are widely used: one uses excess hormone and a limited amount of antibody and is referred to as a competitive immunoassay, whereas the other uses excess antibody and is called either a noncompetitive or immunometric assay. For quantification purposes, immunoassay methods require a labeled marker, which previously was a radioactive [iodine-125 ( ¹²⁵ I)] marker in RIAs and IRMAs but now an enzymatic or a chemiluminescent marker is used in current immunoassays. A detailed description of the principles pertaining to the two major types of immunoassay methodologies is given below. Since RIAs and IRMAs are rarely used now, the focus will be on competitive and immunometric assays using enzymatic or chemiluminescent markers.

Principle of Competitive Immunoassays

The principle of an antigen-excess type of hormone immunoassay involves competition between the hormone being measured and the labeled form of the hormone for a limited amount of the antibody prepared against the hormone ( Fig. 32.1 ). The hormone can be a protein, peptide, or small-molecule. When all three components are combined in a test tube, the net result is a mixture of labeled and unlabeled hormone bound to the antibody and unbound labeled and unlabeled hormone. From the practical standpoint in the assay, the bound hormone is separated from the unbound hormone and the activity of the bound fraction is quantified. Separation of hormone-bound and unbound antibodies is achieved by one of a variety of different methods, including the use of a second antibody (prepared against the first antibody), which precipitates the antigen-antibody complex, or by adding an adsorbent such as activated silica gel particles to bind free antigen. In this manner, by using different concentrations of a pure hormone (standard, calibrator), one can first generate a standard curve of the hormone. As the concentration of the standard is increased, the antibody-bound labeled marker is displaced; the higher the amount of standard that is added, the lower the amount of marker will be obtained. Thus, in an antigen-excess immunoassay, the standard curve shows an inverse relationship between the different amounts of antibody-bound labeled antigen and the different concentrations of the standard ( Fig. 32.2 ). A standard curve is essential in any immunoassay method used to measure the levels of a hormone in a biological fluid.

Fig. 32.1, Measurement of hormones by antigen excess radioimmunoassay: theoretical considerations.

Fig. 32.2, Measurement of hormones by antigen excess radioimmunoassay: practical considerations.

Measurement of a hormone in serum, plasma, or urine by a competitive immunoassay is obtained by first determining the amount of the hormone that is antibody-bound in the sample. The concentration of the hormone is then extrapolated off the standard curve, as shown in Fig. 32.3 . An appropriate computer program can be used to generate the concentrations rapidly. A detailed description of the major components of immunoassay systems is given below.

Antibody

The antibody is perhaps the most important component of an immunoassay method. If an antibody in an immunoassay recognizes only the compound that it is intended to measure, then it is likely that the assay will be not only highly specific but also highly accurate.

Antibodies are commonly produced in animals (polyclonal) or via cell culture (monoclonal). The typical protocol for production of polyclonal antibodies is to disperse a small amount of antigen into an adjuvant (e.g., Freund adjuvant) and to inject it intradermally at multiple sites into an animal such as the rabbit. After approximately 3 months, a blood (serum) sample is obtained from the animal and the antibody titer is determined. This procedure involves the addition of a fixed amount of labeled antigen to serially diluted aliquots of the serum obtained from the animal. The antibody dilutions typically start at 1:1000 and cover a range of at least two orders of magnitude. After the bound antigen is separated from the unbound antigen, the dilution at which the antibody binds between 30% and 70% (usually 40%–50%) of the total activity of the labeled antigen is used for the assay.

To obtain a monoclonal antibody, it is first essential to inject the antigen into a mouse to induce an immunological reaction in the spleen. Each immunized spleen cell can produce an antibody with specific characteristics. The most important step in the production of monoclonal antibodies is screening the spleen cells to separate those capable of secreting a single antibody type. When a myeloma cell from the same species of mouse is fused with the selected spleen cell, a hybridoma is produced. This hybridoma continuously secretes antibodies with the same characteristics as the selected spleen cell. A hybridoma cell is capable of producing hundreds of specific antibody molecules per second. Thus, a continuous clone line can be maintained in culture, becoming a source for the production of homogeneous monoclonal antibody molecules.

Antigen

Here the term antigen can refer to the substance that is injected into an animal to produce an antibody or to the standard that is used to generate a standard curve in an immunoassay. In addition, antigen can refer to the substance that is being measured in a biological fluid.

Antigen Used to Prepare an Antibody

In general, any molecule larger than 10,000 Da can elicit an antibody response. With molecules in the 1000- to 10,000-Da range, eliciting an immune response becomes more difficult. Molecules smaller than 5000 Da are generally nonantigenic and are coupled to a large protein molecule (e.g., albumin); these small molecules are referred to as haptens. Thus, steroids and some peptides, but not proteins, are prepared as hapten-proteins and injected into the animal to produce a polyclonal or monoclonal antibody.

The site of attachment of the protein carrier (e.g., albumin) is important because it determines the specificity of the antibody. In general, highly specific antibodies are formed when the attachment site does not involve any functional group on the steroid molecule. A commonly used approach is to prepare a carboxyl derivative at the carbon-6 position of the steroid molecule and to couple it to an amino group of the protein carrier.

Antigen Used as Standard

Steroids and peptides are available commercially and in most instances can now be obtained in a relatively pure state. However, it should never be assumed that these products are 100% pure when developing an immunoassay. If necessary, the compounds can be purified (e.g., by use of high-performance liquid chromatography [HPLC]).

Unlike steroids and peptides (e.g., GnRH), proteins are considerably more difficult to purify. Although some proteins such as insulin are available in a highly purified state for use as standards in immunoassays, others such as FSH, LH, and hCG have not yet been prepared in sufficient amounts in a highly purified form. Although the latter proteins are available from international agencies, their purity, based on biological potency per unit weight, is often less than that of highly purified preparations reported by individual investigators. To be able to compare results obtained in different laboratories and at different times in the same laboratory, considerable effort has been made to use a single material as a standard.

Reference materials are available for FSH, LH, and hCG. They are provided by the World Health Organization (WHO), which obtains them from the National Institute for Biological Standards and Controls in Hertfordshire, England. Two types of reference materials are available: namely, an International Standard (IS) and an International Reference Preparation (IRP). The IS is a material that has a potency established in 10 to 20 expert laboratories throughout the world, whereas the potency of an IRP is established by only several laboratories.

With the advent of RIAs for gonadotropins, a partially purified extract of human pituitary glands (code name LER-907) was made available by the National Institutes of Health in the United States. This preparation was provided in large amounts to the WHO, which purified it further in 1976 and called it the First International Reference Preparation of Pituitary FSH and LH (1st IRP-FSH and LH). Subsequently, another partially purified extract of LER-907 was prepared in 1980 to replace the 1st IRP-FSH and LH. It was called the Second International Reference Preparation of Pituitary FSH and LH (2nd IRP-FSH and LH) and was assigned the code number 78/549. Highly purified preparations of pituitary FSH and LH have also been prepared. The First International Reference Preparation for Human Pituitary LH (1st IRP-LH, code number 68/40) was available in 1974. Subsequently (in 1988), this material was replaced by the Second International Standard for Pituitary LH (2nd IS-LH; code number 80/552). In 1986, the First International Standard for Pituitary FSH (1st IS-FSH, code number 83/575) was established. Presently, both the 2nd IRP-FSH and LH and 2nd IS-LH are used as standards in gonadotropin immunoassays.

The initial standard used in hCG immunoassays was a partially purified hCG preparation obtained from urine of women in their first trimester of pregnancy and was called the Second IS for hCG. Recently, a highly purified preparation was developed and was referred to as the Fourth IS. This preparation is presently used as the standard in hCG immunoassays. Current standards used for the prolactin and thyroid-stimulating hormone (TSH) immunoassays include the Third IRP WHO Reference Standard 84/500 and Second IRP WHO Reference Standard 80/568, respectively.

Labeled Antigen

Use of an optimum labeled antigen is also important in an immunoassay, as this determines how the compound being measured in a biological fluid, such as serum, is quantified in the assay. The labeled antigen must not only be identifiable by some physical or chemical method (e.g., a radioactivity counter or spectrophotometer) but also must bind to the antibody.

The classical labeled antigens for immunoassays have been radioactive antigens. For example, insulin, the first hormone measured by an RIA, was labeled with radioactive iodine ( ¹³¹ I). This isotope has a very short half-life (8 days) and was soon replaced with ¹²⁵ I, which has a half-life of 60 days. Peptides have also been labeled with ¹²⁵ I. For steroids, tritium ( ³ H) was the initial isotope used in RIAs but was subsequently replaced with ¹²⁵ I. It is important to realize that, unlike proteins and peptides, the steroid molecule itself cannot be iodinated. Instead, the steroid molecule is attached chemically to an iodinated carrier molecule such as histamine or tyrosine methyl ester.

Steroid Hormone RIAs

In contrast to the protein and peptide RIAs, the estradiol RIA method developed by Abraham ² involved the separation of estradiol from interfering metabolites by organic solvent extraction and Celite or Sephadex column chromatography, prior to its quantitation by RIA. The purification steps were added to remove most of the estrogen metabolites. Estradiol is readily converted to estrone and both estradiol and estrone are converted to a total of approximately 90 metabolites. The extraction step removes the conjugated (water-soluble) steroids, which represent about two-thirds of the total metabolites, whereas the chromatographic step separates estradiol from potential interfering unconjugated metabolites. The estradiol RIA included a specific antiserum against estradiol in conjunction with tritiated estradiol, and separation of the antibody-bound and unbound estradiol fractions using charcoal. Soon afterward, it was applied successfully to other sex steroid hormones, such as testosterone and progesterone.

A notable change in the RIA method during the 1970s was the replacement of tritium with iodine in the labeled antigen to improve assay sensitivity. Because of their relatively low cost, these RIAs became widely used in diagnostic and research laboratories.

Validation of RIAs

A critical part of assay development is validation of the assay. Before an immunoassay can be used to measure any compound, it must first be validated with respect to specificity, sensitivity, accuracy, and precision. This was done for the RIAs described above. Since these validation parameters are important, they are described in detail below.

Specificity

Assay specificity refers to the degree of interference from cross-reaction that is encountered from substances other than the target analyte. The specificity of an immunoassay is usually assessed in two different ways. First, the cross-reactivity (expressed as a percentage) of the antibody is determined by comparing the dose-response standard curve of the substance being measured with dose-response curves obtained for compounds that may be present in the same sample as the analyte, and that may bind to the antibody. The percent cross-reaction of an antiserum with a substance is calculated from the mass of standard that yields 50% inhibition of binding of the assay marker to the antibody, divided by the mass of the cross-reacting substance that gives the same percentage of inhibition (50%) and multiplied by 100%. Ideally, the cross-reaction should be <0.1%.

A second approach for defining immunoassay specificity is to compare the analyte values of a group of samples measured by an immunoassay method with those obtained by a “gold standard” assay method such as MS. If this is not possible and the analytes are being measured in an immunoassay without chromatography, the analyte values should be compared with the values obtained by an immunoassay that uses an extraction step followed by a chromatographic step (e.g., Celite column partition chromatography) to separate the analyte in question from interfering metabolites.

Sensitivity

The sensitivity of an assay is defined as the smallest amount of the substance being measured that can be distinguished from zero. Two parameters are generally determined to define the sensitivity of an assay, namely the lower limit of quantitation (LLOQ) and the limit of detection (LOD). The LLOQ is the lowest concentration of an analyte in a sample that can be quantitatively determined with suitable accuracy and precision. In contrast, the LOD is the lowest concentration of an analyte that is statistically distinguished from zero, but its value is not sufficiently accurate or precise. In practice, the LLOQ can be determined by assaying 10 replicates of each concentration of standard and the “zero” standard, which contains no standard. This allows calculation of the mean ± standard deviation (SD) amount of the antibody-bound marker corresponding to each concentration of standard. The LLOQ of an assay is the lowest standard concentration that yields a mean amount (e.g., counts per minute) of antibody-bound marker differing by two SDs from the mean amount of antibody-bound marker associated with the zero standard. As for the LOD of an assay, it can be determined by extrapolating the value between the zero standard and the LLOQ.

Sometimes, in assays using one or more purification steps, the assay sensitivity is expressed in terms of the lowest amount of analyte that can be measured per milliliter of sample. This can be determined by calculating the product of the LLOQ value by any dilution factors, as well as the factor accounting for procedural loss (in assays using an extraction and/or chromatographic step).

The sensitivity of an assay is especially important when very low serum levels of a compound are being analyzed (e.g., measurement of estradiol in samples from postmenopausal women or from patients treated with an aromatase inhibitor).

Accuracy

Assay accuracy defines the extent to which a given measurement agrees with the actual value. One commonly used method to establish the accuracy of an immunoassay is based on the finding of linearity (parallelism) between the assay standard curve and serial dilutions (using assay buffer) of several samples containing known high concentrations of the analyte. Another method is based on the recovery of added (“spiked”) standard at different levels from patient samples. This method is often used only for small-molecule assays (e.g., steroids) due to the labile nature of proteins and large peptides. Both the linearity and “spiked” sample methods test for assay interference by determining the proportionate levels of analyte detected when a sample is diluted serially or spiked with different amounts of a standard; a difference of ± 10% at each concentration is generally accepted. However, these methods are limited by the precision of carrying out the dilutions or “spiking.”

Precision

The precision of an assay refers to the variability that exists when multiple measurements of the compound are made on the same sample. In practice, both intraassay precision and interassay precision are determined and are usually expressed as the coefficient of variation (CV) of replicate measures. The CV (expressed as a percentage) is calculated by dividing the SD by the mean of replicate determinations of an analyte and then multiplying by 100. Intraassay precision is assessed by measuring the analyte in replicate samples (usually 7–10) within the same assay. Interassay precision is determined by measuring the analyte in replicate samples (at least five), with each sample included in a different assay. Both intraassay and interassay precision should be determined at three different concentrations of the analyte (high, midrange, and low). In general, the acceptable intraassay and interassay CVs are 10% and 15%, respectively.

Impact of the RIA Method

The immediate impact of the RIA method was that it allowed measurement of an immensely wide range of compounds of clinical and biological importance, and it opened new horizons in endocrinology. The long-term impact of the RIA method was that its use in numerous studies enriched the field of endocrinology with new knowledge, and its use in diagnostic testing provided physicians with valuable information for diagnosing and treating countless patients. The RIA methodology also allowed substantial research into the physiological and pathophysiological roles of hormones in applications such as sexual differentiation, puberty, neuroendocrinology, the menstrual cycle, pregnancy, menopause, and male endocrinology. In addition, the RIA method opened the door for epidemiologic studies that permitted us to better understand the role of hormones in the etiology of numerous diseases, notably hormone-dependent breast and prostate cancers.

Development of Sandwich-Type Immunoassays for Proteins and Peptides

In the early 1970s, RIAs for protein and peptide hormones began to be replaced with antibody-excess types of assays. They are known as sandwich immunoassays because the antigen (analyte) is “sandwiched” between two antibodies, which achieved greater assay sensitivity and specificity. An immunoradiometric assay (IRMA) was developed first and then some years later a midradioactive version of the sandwich assay, called the enzyme-linked immunosorbent assay (ELISA), was developed.

In contrast to antigen-excess immunoassays, which use a limited amount of antibodies resulting in competition between labeled and unlabeled antigens, there is no competition between these antigens in antibody-excess immunoassays (sometimes referred to as immunometric assays). In general, two different antibodies (capture and detection) that recognize two different parts of the antigen are used in antibody excess immunoassays ( Fig. 32.4 ). Thus, this assay method is useful for proteins and large peptide hormones. Quantitation is achieved by labeling the detection antibody with a radioactive ( ¹²⁵ I) or nonradioactive (enzyme) marker for the IRMA and ELISA, respectively. In contrast to the inverse relationship between antibody-bound labeled antigen and concentration of antigen in the standard curve in an antigen-excess immunoassay, there is a direct relationship between the labeled antibody and antigen concentration in an antibody-excess immunoassay ( Fig. 32.5 ).

Fig. 32.4, Principle of the antibody excess immunoassay: theoretical consideration shown for the ELISA.

Fig. 32.5, Nomogram of 17α-hydroxyprogesterone (17α-OHP) in different types of patients with congenital adrenal hyperplasia.

The two-site IRMA and ELISA methods were ideal for use with monoclonal antibodies, thereby increasing not only assay sensitivity but also assay specificity. In addition, two-site assays offer greater sensitivity because they are much less dependent on the affinity of the antibody than RIAs. These assays opened up new clinical diagnostic opportunities.

The ELISA differs from the IRMA in that the ELISA uses a solid-phase procedure and an enzyme-labeled antibody instead of a radioactively labeled antibody. The most common format used for the ELISA is the multi-well plate made of polystyrene or polyvinyl. This is convenient to handle since centrifugation is not required and multiple wash steps are readily automated. Antibody is coated on the wall of each well and this antibody binds to the corresponding antigen in the test sample. The antigen-antibody complex is quantified by the addition of an enzyme-labeled specific antibody. Following the addition of the appropriate substrate, the endpoint (product) can be “read” spectrophotometrically using an automated reader.

Advantages and Limitations of Indirect Steroid RIAs

Steroid RIA methods with purification steps have the following advantages: First, steroid-binding proteins (e.g., sex hormone-binding globulin [SHBG]) are denatured, thereby releasing the steroids (e.g., estradiol and testosterone) that they bind. Second, the purification steps remove numerous potentially interfering metabolites, prior to RIA. Third, the RIAs are accurate and reliable when properly validated. Finally, multiple steroids (usually up to five) can be measured in a single aliquot of serum.

Indirect steroid RIAs also have limitations. They are cumbersome, time-consuming, costly, and require relatively large sample volumes, especially when the steroid is present in low concentrations. Also, although multiple steroids can be measured in a single aliquot of serum, the measurements have to be done very carefully and are especially time-consuming. In addition, as with all antibody-based assays, since the measurement of the analyte is a surrogate approach (i.e., radioactivity is measured rather than the actual analyte itself), there is always the possibility of antibody cross-reactivity giving an erroneous result. Furthermore, the presence of autoantibodies within patients can further affect an assay, leading to falsely high or low values depending on the type of antibody interaction that occurs.

Development of Direct Steroid Immunoassays

Due to the time-consuming limitations of indirect RIAs, the organic solvent extraction and chromatography steps used prior to RIA were eliminated in the late 1970s, allowing direct RIAs to be performed. This resulted in rapid measurements of steroid hormones. Indirect RIAs continued to be used, but their use was overwhelmingly surpassed by direct RIAs, particularly in clinical diagnostic laboratories.

Direct RIAs have the advantage of being convenient, simple, rapid, and relatively inexpensive, and requiring a lower sample volume (usually 0.1 mL). However, these assays also have serious limitations. They often overestimate the measurements due to a lack of specificity of the antibody. Overestimation of testosterone and estradiol levels in serum by direct immunoassays has been demonstrated in a substantial number of studies. ^, The overestimation is especially evident in samples obtained from women treated with exogenous steroid hormones. Also, matrix differences may exist between serum samples (particularly hemolyzed and lipemic samples) and solutions of the standard used to prepare the standard curve in the assay. In addition, steroids such as testosterone and estradiol may not be released efficiently from proteins such as SHBG to which they bind with high affinity in blood. Furthermore, direct RIAs generally lack the sensitivity to measure accurately low levels of certain steroid hormones such as estradiol and testosterone. Finally, these assays generally measure only a single analyte at a time. Limitations of direct immunoassays for quantifying estradiol in postmenopausal women and testosterone in both premenopausal and postmenopausal women are now well documented in the literature.

Development of Commercial Immunoassay Kits

In the early 1980s, commercial IRMA kits were developed to measure proteins and peptides as well as direct RIA kits to measure steroids and other small-molecule hormones. Soon afterward, midradioactive forms of these assays were developed. Using the commercial kits, protein and peptide hormones were then measured manually by ELISA (antibody excess) and steroid hormones by a competitive EIA (antigen excess). Subsequently, both assay methods were automated after replacing the enzyme marker with a chemiluminescent or fluorescent marker.

Automated Immunoassays

Over the past 30 years or so, highly sophisticated automated immunoassay systems have been developed. These systems consist of instruments that not only detect spectral properties of nonradioactive ligands (e.g., chemiluminescence) but can also analyze multiple analytes and process multiple samples rapidly and efficiently. These immunoassay systems are capable of using both antigen-excess and antibody-excess methodology.

Currently, a widely used immunoassay system is the Immulite analyzer (Siemens Healthcare Diagnostics, Deerfield, Illinois). The Immulite system employs enzyme-amplified chemiluminescent technology. The mechanism involves hydrolysis of a stable chemiluminescent substrate through the action of the enzyme alkaline phosphatase, resulting in an unstable anion that gives rise to sustained emission of light. The emitted light is quantified by the use of a luminometer in the instrument.

As an example, the Immulite luteinizing hormone (LH) assay uses a solid-phase, two-site immunochemiluminometric assay (ICMA). The solid phase consists of a polystyrene bead coated with a monoclonal antibody against LH. The bead is sealed into an Immulite test unit, to which LH standard or serum sample, together with a polyclonal antibody conjugated to alkaline phosphatase, are added simultaneously. During an incubation period, LH is bound to the monoclonal antibody coating the bead and a polyclonal antibody-enzyme conjugate is added, forming a “sandwich complex.” After unbound conjugated antibody is removed by a wash, the amount of complex (which is directly proportional to the LH standard or LH in the sample) is quantified by the use of the chemiluminescent substrate described previously.

Interferences in Immunoassays

Biotin

Biotin (vitamin B7) is a water-soluble vitamin that is involved in many enzymatic activities that regulate the metabolism of fat, carbohydrates, and amino acids. It is not made in the body but is available in many plant- and animal-based sources of food. Biotin is available in over-the-counter multivitamin preparations and has been used increasingly as a supplement to enhance skin, nail, and hair health, even though there is a lack of evidence to support this use.

Biotin forms an irreversible complex with a variety of proteins. This interaction is widely used to anchor capture antibodies to the solid phase of immunoassays. This can be achieved by preparing a biotinylated antibody and coating magnetic beads with streptavidin, which has a high affinity for biotin. The mixture forms a streptavidin-biotin-capture antibody complex, to which an analyte binds. A detection antibody is then added to complete the “sandwich.” In the presence of high biotin concentrations, biotin saturates the streptavidin binding sites and alters the attachment between the biotinylated capture antibody and streptavidin, resulting in a low signal and falsely low concentrations of the analyte.

In competitive immunoassays that have the streptavidin-biotin-antibody complex attached to a magnetic bead, high concentrations of biotin will also saturate the streptavidin resulting in a low signal. However, in contrast to the sandwich-type assay in which a falsely low concentration of analyte is obtained, a competitive assay will give rise to a falsely high concentration of analyte because the signal is inversely proportional to the analyte concentration.

Heterophilic Antibodies

Circulating human antibodies that react with animal proteins (anti-animal antibodies) are often an unrecognized and unsuspected source of interference in immunoassays. This is particularly true with two-site immunoassays. The most common human anti-animal antibody interference is caused by human antimouse antibodies (HAMA). HAMA can cause either positive or negative interference in two-site mouse monoclonal antibody-based immunoassays. When interference is suspected, samples can be retested with a different immunoassay since some immunoassays have been developed to minimize the effects of HAMA. Analyzing the sample with heterophilic antibody-blocking reagents or diluting the sample and testing the linearity can also be done.

Autoimmune Antibodies

Autoantibodies may interfere in certain immunoassays. The best-known example is the thyroglobulin immunoassay, in which antithyroglobulin antibodies cause falsely low thyroglobulin measurements. These autoantibodies can be found in healthy individuals and in thyroid cancer patients. The interference problem can be resolved by measuring thyroglobulin using MS.

Hook Effect

The hook effect can occur in sandwich-type immunoassays when a hormone at very high concentrations saturates both the capture and detection antibodies, preventing formation of the sandwich. As a result, the detected signal (e.g., flashes of light in a chemiluminescent immunoassay) will indicate low or mildly elevated concentrations. The name “hook effect” is based on the phenomenon that occurs with exceedingly high concentrations of an analyte (i.e., an initial increase in binding of the analyte to the antibodies). At some critical point, the binding starts to hook down. The hook effect is important clinically (such as in cases of prolactinoma). With exceedingly elevated levels of prolactin, a false report of only mildly elevated levels may result in an erroneous diagnosis of a midfunctioning pituitary tumor and subject the patient to unnecessary surgery. Other testes susceptible to the hook effect include β-hCG in patients with choriocarcinoma and thyroglobulin in thyroid cancer. One way to establish the true hormone concentration when the hook effect is suspected is to perform sample dilutions of 1:100 and more prior to measuring the hormone.

Mass Spectrometry Assays

Measurements of steroid hormones by MS assays actually preceded their measurements by RIA. As early as 1966, the first comprehensive urinary steroid profile was produced by gas chromatography-mass spectrometry (GC-MS). This method combines the resolving power of gas chromatography with the high sensitivity and specificity of the MS. The separation of steroids by gas chromatography requires that they be first derivatized to increase their volatility, selectivity, and detectability. The MS functions as a unique detector that provides structural information on individual solutes as they elute from the gas chromatography column. The MS technique first involves ionization of the compound being measured at the ionization source and is followed by separation and detection of the ions in the mass analyzer. A mass spectrum is produced in which the relative abundance of a particular ion is plotted as a function of the mass-to-charge (m/z) ratio, and the concentration of the compound is then obtained.

Due to the complexity of the methodology and associated costs of the instrumentation and reagents, as well as the need for a highly trained individual to carry out the assays, the use of GC-MS assays was restricted to a limited number of laboratories, primarily in pharmaceutical companies. Thus, for a period of about 30 years after the development of the first RIA in 1969, conventional RIAs and direct immunoassays were the predominant methods used to quantify steroid hormones in clinical diagnostic and research laboratories due to their relative ease of performance and considerably lower overall costs. However, advances in liquid chromatography (LC) technology in the 1980s led to the development of a high-performance liquid chromatography-MS (LC-MS) instrument in 1987. In addition, the invention of an electrospray source by Nobel laureate John B. Fenn in 1990, and the subsequent development of chemical ionization, greatly improved routine analysis of steroids. This technology facilitates ionization of compounds present in liquid droplets and sprays the molecules directly into the MS from the HPLC unit. The advancements allowed for simple coupling of the LC eluent with the MS and often negated the need for derivatizing the steroid, thereby reducing the complexity of the assay and shortening the assay run time dramatically. These factors greatly increased the throughput of patient samples while still providing highly accurate and precise results.

In recent years, there has been a large increase in the use of assays that use either LC or gas chromatography with tandem MS (LC-MS/MS or GC-MS/MS). Tandem MS consists of two MS in series connected by a chamber (collision cell). After chromatography, the sample is processed in the first MS to obtain the precursor ion, which is then fragmented in the collision cell into product ions. The mass of the product ions is then determined in the detector of the second MS. This method has high specificity, sensitivity, and throughput.

During the past decade, there has been a substantial increase in the use of LC-MS/MS assays, particularly in major diagnostic clinical laboratories, and to a lesser extent in some research laboratories. However, there are still situations where a GC-MS assay provides higher chromatographic resolution and even sensitivity than LC-MS. A particular strength of GC-MS and GC-MS/MS assays is their high applicability to the measurement of large numbers of structurally similar compounds. They remain the most powerful assay method for defining defects in steroid hormone metabolism.

Because of the high validity and throughput of steroid hormone MS assays, there is a rapidly growing use of this methodology in both clinical and research laboratories. In larger reference laboratories, these assays have replaced the indirect RIAs, which are cumbersome and time-consuming, and direct immunoassays, which lack specificity and/or sensitivity. The MS technology has been implemented successfully for routine analysis of steroid hormones in major clinical diagnostic laboratories. Although the high cost of MS instrumentation, related operating costs, and requirement for high technical expertise have prohibited smaller laboratories from using this instrumentation for high-throughput routine testing of steroid hormones, this situation is changing and MS assays are becoming much more widely used.

In addition to the use of MS methodology for routine analysis of steroid hormones, this methodology is now sufficiently rapid and robust for measuring these hormones in large epidemiological studies with high specificity and sensitivity. An important advantage of MS assays is their capability of measuring multiple steroids in a single aliquot of serum or urine. In contrast, generally only up to five different steroids can be measured in a single serum aliquot (usually 1 mL) by indirect RIA. At the National Cancer Institute, LC-MS/MS assays were developed for quantifying as many as 15 different estrogens in only 0.5 mL of serum or urine. ^, In addition, LC-MS/MS assays have been established for simultaneous quantitation of 11 androgens, including principal adrenal and gonadal androgenic precursors and their 5α-reduced metabolites.

Studies using MS have shown that the adrenal steroid, 11β-hydroxyandrostenedione, is a precursor to 11-ketotestosterone and 11-keto-5α-dihydrotestosterone, which are potent androgenic androgens. In addition, several 11-oxygenated androgens (11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone, and 11-ketotestosterone) have been found to be elevated in women with polycystic ovary syndrome (PCOS) and cumulatively constitute a greater proportion of total circulating androgens than DHEA, androstenedione, and testosterone. More work on the clinical significance of these MS-based 11-oxygenated androgens is clearly needed.

Measurement of metabolite profiles of a steroid hormone has the potential to provide highly valuable information in diagnosing patients and in a variety of studies, particularly epidemiological studies. Quantitative analysis of metabolite profiles of small-molecular-weight molecules such as steroid hormones is referred to as metabolomics and is a rapidly growing field of research.

Strengths and Limitations of Mass Spectrometry Assays

Strengths

A notable strength of MS assays is that they are highly specific and sensitive; this is especially evident in steroid hormone measurements. Another very important strength is that multiplexing can be used with MS assays. This allows measurement of multiple compounds in the same sample, which can be used to obtain profiles of principal steroid hormones and their metabolites. Comprehensive analysis of steroid profiles may potentially improve patient outcomes. MS applications in the analyses of comprehensive steroid panels have been demonstrated in conditions such as congenital adrenal hyperplasia (CAH) in children and in predicting metabolic risk in patients with polycystic ovary syndrome. MS methodology has also demonstrated the capability to detect proteins and peptides in a specific and sensitive manner. This methodology is expected to overcome some of the limitations of protein immunoassays that were discussed earlier.

Limitations

Although MS assays are reported to be the gold standard for steroid hormone measurements, they have some limitations that include the following: (1) development of optimal internal standards is not easy to achieve; (2) a highly trained technician is required to operate the MS instrument, and the instrument and associated items (e.g., reagents, supplies, and maintenance contracts) are expensive; (3) the overall cost prohibits use of an MS in small laboratories; (4) finally, for diagnostic testing, MS assays are used mostly to measure small-molecule hormones, particularly steroid hormones, and not reproductive protein hormones such as LH, FSH, hCG, and prolactin.

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